EP1582214A2 - Use of chondroitin mono- and disulphates in therapy - Google Patents
Use of chondroitin mono- and disulphates in therapy Download PDFInfo
- Publication number
- EP1582214A2 EP1582214A2 EP05290624A EP05290624A EP1582214A2 EP 1582214 A2 EP1582214 A2 EP 1582214A2 EP 05290624 A EP05290624 A EP 05290624A EP 05290624 A EP05290624 A EP 05290624A EP 1582214 A2 EP1582214 A2 EP 1582214A2
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- EP
- European Patent Office
- Prior art keywords
- chondroitin
- cells
- use according
- sulfuric
- sulfate
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
Definitions
- the present invention is directed to the field of the necessities of life, and more particularly in the field of biological chemistry.
- chondroitins consisting primarily, but not exclusively, of disaccharide sulphated at 2,4,6-position and / or 2,6-disulfated disaccharides; and able include non-sulphated disaccharides, as factors of conservation and regeneration of cartilage.
- chondroitins are obtained by enzymatic cleavage of Proteinoglycans already sulfated, contained in the cartilages of different species animal, and in particular of bovine, porcine or ichthyic origin.
- the chondroitin sulphates are in the form of long chain polysaccharides where they are associated with an axial protein, itself linked to sulphated keratans, and the whole is known as aggrecan.
- Enzymatic cleavage can be performed by a chondroitinase and chondroitinase C or chondroitinase ABC which provides including 6-sulfuric, 4-sulfuric and 2,6-disulphuric disaccharide, accompanied by more sulphated products. Non-sulphated disaccharides present in all origins are also released by this enzymatic digestion.
- the analysis of the chondroitin sulphates can be carried out by NMR and shows different spectra which are the consequence of the presence of the different sulphatations and of their respective proportion.
- the FTIR analysis shows in the region between 1000 and 500 cm -1 , and more particularly between 950 and 700 cm -1 , characteristic peaks of each of the chondroitin sulfuric acids.
- the various chondroitins of porcine, ichthyic (shark) and bovine origin have different spectra which attest to the presence and proportions of different sulfuric disaccharides in different quantities.
- a ⁇ -elimination reaction (between xylose and serine), performed on chondroitin sulfates after extraction, used to evaluate the rate and mass protein that would remain bound despite purification processes.
- the analytical technique used here is liquid chromatography on an exclusion column with as a detector but not exclusively, a refractive index detector. This method provides information on the presence of bound residual proteins.
- the quantity of 6-sulfuric derivatives obtained is dependent as for 4 sulfuric derivatives, animal origin and extraction tissue. So for all origins, apart from the shark the values are always lower than those of the 4-sulfuric derivative.
- sulfuric derivatives represent at least 40% of the all disaccharides.
- chondroitin 2,6-disulphuric acid is concentrations greater than 10% than in mixtures of fish origin.
- Chondroitin sulfuric or disulfuric acids resulting from cleavage enzymatic cartilage tissue plays an important role in physical properties and chemical cartilage. At high concentrations they attract water into the tissues of so that the collagen is put under tension, which in turn contributes to the resistance mechanical cartilage to compressive forces. These molecules synthesized by chondrocytes and secreted in the intercellular space, are related to hyaluronans and form molecular complexes of high molecular weight.
- the articular chondrocytes are responsible for the slow evolution of the extracellular matrix, which constitutes one of the causes of tissue damage.
- Polysulfated polysaccharides and in particular those consisting of Disulfated disaccharides have been used in patients with osteoarthritis and have showed significant therapeutic results, especially by slowing down progression of the disease.
- Mono and / or disulfated disaccharides can be administered by and can reach sufficient concentrations in body fluids - like the synovial fluid- and in the joints to exert beneficial effects.
- [ 35 S] -labeled molecules has shown significant serum concentrations, and in particular in animals where there is a selective tropism towards GAG-rich tissues, such as intervertebral discs, the eyes and cartilage of the joints.
- GAG-rich tissues such as intervertebral discs, the eyes and cartilage of the joints.
- the mono- or disulfated chondroitin acids exhibit a tropism for the knee joint, as evidenced by scan (scanning) of the bone by 99 Tc-labeled CS.
- Chondroitin mono- or disulphuric acids have an effect protector on cartilage damage.
- Chondroitin mono- or disulfurics manifest "in vitro" increases in the synthesis rate of aggrecans on human chondrocytes and thereby help to increase the matrix proteins, mainly PGs, in the immediate environment of chondrocytes, and thus prevent their dedifferentiation.
- chondroitins 2,4- or 6-sulfuric disaccharides as well as 2,6-disulphuric acid increased production and aggregation ability of proteoglycans (PG) newly synthesized that accumulate all around the chondrocytes put in incubation under basic conditions.
- PG proteoglycans
- monounsaturated or disulphuric chondroitin have the effect of at least partially reversing the IL-1 induced disintegration of proteoglycans, and this effect is observed "in vitro" even in the absence of any intervention on cells. This effect does not alter the chondrocyte phenotype as it is demonstrated by stable expression of type II collagen and aggrecan, without hyper-regulation of type I collagen.
- This effect is not related to sulfation of the molecule because heparin is inactive to increase proteoglycan synthesis.
- chondroitin acids increase the proportion of newly synthesized PGs that accumulate in the intercellular space. This presence of PG in large quantities in the chondrocyte environment is key to ensuring the preservation of the chondrocyte phenotype and the physico-mechanical properties of the cartilage.
- chondroitin mono- or disulphuric acids can find an explanation in an increase in the synthesis of filaments more of hyaluronan, in the amount of hyaluronic acid synthesized and in the inhibition enzymatic activities, such as elastase and aggrecanase or cathepsin B. It can therefore be assumed that monounsaturated or disulphuric chondroitin have an effect on the regulation of metalloprotease actions induced by IL-1 ⁇ and thus preserve the stock of non-degraded PGs.
- chondroitin sulfuric acid extracted from porcine cartilage exhibits potent aggregating power with an increase in high molecular weight aggregates (AHMW) of greater than 80% as measured by total radioactivity, whereas cartilage extracted acids other animal species, such as chicken or shark, gave 70% increase in reaggregation in the presence of interleukin IL-1 ⁇ compared to the normal state.
- AHMW high molecular weight aggregates
- Chondroitin mono- or disulfuric acids of the invention increase the percentage of functional sulfated macromolecules in the direct environment of chondrocytes "in vitro", mainly by showing aggregation properties of PGs.
- the Peri-cellular retention of bulky aggrecans after induction by the acids mono- or disulphuric chondroitin of the invention, plays a favorable role in retarding, or better to suppress, the process of dedifferentiation and to bring about a recovery of a differentiated phenotype.
- Mono (2,4 or 6) sulfated chondroitin acids and chondroitin acid 2,6-disulfuric acid of the invention are used for therapeutic purposes, particularly by oral or parenteral, in the form of pharmaceutical compositions such as tablets, dragees, capsules, pills, cachets, powders, flavored or not, or injections intended for intramuscular and intra-articular, in the form of ampoules, single-dose vials or self-injecting syringe.
- a usual dose for the oral or parenteral route ranges from 0.2 g to 2 g per unit dose and preferably 0.4 g to 0.6 g per dose.
- Cartilaginous tissue in this case the pieces of cattle trachea or pigs, is chopped into small pieces and subjected to enzymatic hydrolysis in using as the proteolytic enzyme the enzyme marketed under the name Prolyve 100® by the company LYVEN. It is a liquid alkaline protease, obtained at from a selected strain of Bacillus licheniformis. Fermentation is oriented to produce essentially an alkaline protease of the serine type, also called “Subtilysin Carlsberg” (EC 3.4.21.14).
- Chondroitins are separated after filtration by addition of ethanol with stirring.
- the precipitate is filtered off and then washed with a water / ethanol mixture.
- the precipitate is taken up in a water / sodium acetate mixture and then re-precipitated with ethanol under stirring and then drained, dried in an oven, milled and sieved on a mesh of 450 microns.
- the chondroitin thus obtained is analyzed by proton NMR, which characterizes two very sharp peaks at 2.04 ppm (6S) and 2.02 ppm (4S).
- the KBR pellet FTIR shows between 1500 and 1000 cm -1 the characteristic peaks of chondroitins of different origins (see Figure 2).
- Joint chondrocytes were cultured at high density for 6 days. The cells are incubated in the presence or absence of 10-fold effectors. or 100 ⁇ g / ml in the presence or absence of IL-1 ⁇ for 20 hours. A unique transcript 8.9 kb RNA and a single 5 kb procollagen II ⁇ 1 transcript were observed under basic conditions.
- CS chondroitin sulfuric acid
- CSs on PG synthesis was investigated by incorporation of ( 35 SO 4 ) in chondrocyte cultures treated with CS.Chondrocytes were cultured as described above. Effector was performed in radio-labeled sulphate-free sulfate-free DMEM (DULBECCO Modified Eagle Medium) medium.In the basic conditions, without any effector, the total PG sulfate ( 35 SO 4 ) level, extracted from culture medium (M), and the corresponding cell pool (C) was on average ( ⁇ SEM) of 3.42 dpm x 10 -4 / 10 ⁇ g of DNA with 36% recovered in the cell layer.
- DMEM radio-labeled sulphate-free sulfate-free DMEM (DULBECCO Modified Eagle Medium) medium.In the basic conditions, without any effector, the total PG sulfate ( 35 SO 4 ) level, extracted from culture medium (M), and the corresponding cell pool (C) was on average ( ⁇ SEM) of 3.42
- the action of CSs on the aggregation of neo-synthesized PGs has been investigated.
- the IL 1 ⁇ has the property of disintegrating the PGs, which results when a separation by exclusion chromatography of the PGs synthesized by the chondrocytes is carried out, by an increase in complexes of lower molecular weight.
- AHMW high molecular weight
- CSs are therefore able to limit the disaggregation of complex PG of high molecular weight.
- the amount of aggregated PG eluted between Kav 0.10 - 0.40 was quantified and expressed as a percentage of total radioactivity eluted on the column.
Abstract
Description
La présente invention s'adresse au domaine des nécessités de la vie, et plus particulièrement au domaine de la chimie biologique.The present invention is directed to the field of the necessities of life, and more particularly in the field of biological chemistry.
Elle a plus particulièrement pour objet l'utilisation en thérapeutique de composés dérivés de chondroïtines sulfuriques, extraits de cartilages d'origines diverses.It more particularly relates to the therapeutic use of compounds derived from sulfuric chondroitin, extracts of cartilage of various origins.
Elle a spécifiquement pour objet l'utilisation en thérapeutique des chondroïtines constituées essentiellement, mais non exclusivement, de disaccharide sulfatés en position 2, 4, 6 et/ou de disaccharides di-sulfatés en position 2,6; et pouvant inclure des disaccharides non sulfatés, en tant que facteurs de conservation et de régénération des cartilages.Its specific purpose is the use in therapeutics of chondroitins consisting primarily, but not exclusively, of disaccharide sulphated at 2,4,6-position and / or 2,6-disulfated disaccharides; and able include non-sulphated disaccharides, as factors of conservation and regeneration of cartilage.
Ces chondroïtines sont obtenues par clivage enzymatique des protéinoglycanes déjà sulfatées, contenus dans les cartilages de différentes espèces animales, et notamment d'origine bovine, porcine ou ichtyque. Dans ces cartilages, les chondroïtines sulfates se présentent sous forme de polysaccharides à longue chaíne où ils sont associés à une protéine axiale, elle-même liée à des keratanes sulfates, et l'ensemble est connu sous le nom d'aggrecane.These chondroitins are obtained by enzymatic cleavage of Proteinoglycans already sulfated, contained in the cartilages of different species animal, and in particular of bovine, porcine or ichthyic origin. In these cartilages, the chondroitin sulphates are in the form of long chain polysaccharides where they are associated with an axial protein, itself linked to sulphated keratans, and the whole is known as aggrecan.
Le clivage enzymatique, notamment sous l'action de chondroïtinase ABC, a pour effet de fournir un mélange de disaccharides que l'on appelle :
- d'acide chondroïtine 6-sulfurique,
- d'acide chondroïtine 4-sulfurique,
- d'acide chondroïtine disulfurique, présentant un sulfate en 2 de l'acide glucuronique et un sulfate en 6 de la N-acétyl galactosamine. Selon l'origine du cartilage, les pourcentages en ces différents disaccharides varient. Les tissus cartilagineux d'origine ichtyque fournissent des pourcentages plus élevés en acide 2,6-chondroïtine disulfurique et en acide chondroïtine 6-sulfurique. Au contraire, les trachées d'origine porcine, fournissent des chondroïtines plus riches en acide chondroïtine 4-sulfurique (environ 60 %). Les tissus cartilagineux d'origine bovine fournissent un mélange d'acides chondroïtine 4-sulfurique et 6-sulfurique. Le mélange est souvent accompagné de la présence de molécules non sulfatées.Selon N. Volpi Current Drug Target (2004) 4, 119-127, les chondroïtines sulfates constituent des polyanions et certaines de leurs propriétés sont liées à leur forte charge qui est capable d'attirer l'eau dans les tissus et de les hydrater. Les chaínes d'acides chondroïtine sulfuriques sont synthétisées par des cellules liées d'une manière covalente aux protéines qui sont sécrétées dans la matrice extracellulaire, sous forme de protéoglycanes. Plusieurs familles de protéoglycanes, portant des chaínes d'acides chondroïtine sulfuriques, ont été caractérisées, y compris deux des familles les plus importantes de protéoglycanes trouvées dans la matrice extracellulaire des tissus connectifs : la famille des aggrécans et la famille des protéoglycanes riches en motifs répétitifs leucine.
- La famille des aggrécans est constituée de fractions de haut poids moléculaire (>500 kDa) qui s'agrègent à l'extérieur de la cellule en se liant à l'acide hyaluronique. Elles jouent un rôle important dans les propriétés physiques et chimiques du cartilage.
- of chondroitin 6-sulfuric acid,
- 4-sulfuric chondroitin acid,
- of chondroitin disulfuric acid, having a sulfate in 2 of glucuronic acid and a sulfate in 6 of N-acetyl galactosamine. Depending on the origin of the cartilage, the percentages of these different disaccharides vary. The cartilaginous tissues of fish origin provide higher percentages of 2,6-chondroitin disulfuric acid and 6-sulfuric chondroitin acid. In contrast, tracheal pigs provide chondroitin rich in 4-sulfuric chondroitin acid (about 60%). The cartilaginous tissues of bovine origin provide a mixture of chondroitin 4-sulfuric and 6-sulfuric acids. The mixture is often accompanied by the presence of non-sulfated molecules. According to N. Volpi Current Drug Target (2004) 4 , 119-127, chondroitin sulphates are polyanions and some of their properties are related to their high charge which is capable of attract water to the tissues and hydrate them. Chondroitin sulfuric acid chains are synthesized by cells covalently bound to proteins that are secreted into the extracellular matrix as proteoglycans. Several families of proteoglycans carrying chains of chondroitin sulfuric acids have been characterized, including two of the most important families of proteoglycans found in the extracellular matrix of connective tissues: the family of aggrecans and the family of proteoglycans rich in repetitive patterns. leucine.
- The aggrecan family consists of fractions of high molecular weight (> 500 kDa) which aggregate outside the cell by binding to hyaluronic acid. They play an important role in the physical and chemical properties of cartilage.
Sans vouloir être lié par des considérations d'ordre théorique, il apparaít que, dans les molécules d'aggrecan et autres protéoglycanes, les acides chondroïtine sulfuriques existent sous forme de longues chaínes dont le motif est l'acide D-glucuronique liée par une liaison (1→3) à un N-acétylgalactosamine. Plusieurs types de sulfatation sont observées : pour la N-acétylgalactosamine les sulfatations se retrouvent préférentiellement en 4 et/ou en 6 et en position 2 pour l'acide D-glucuronique.Without wishing to be bound by theoretical considerations, it appears that in aggrecan molecules and other proteoglycans, chondroitin acids sulfuric acids exist in the form of long chains whose motive is D-glucuronic acid bound by a (1 → 3) bond to N-acetylgalactosamine. Several types of sulfation are observed: for N-acetylgalactosamine, sulfations are preferentially at 4 and / or 6 and at position 2 for D-glucuronic acid.
Le clivage enzymatique peut être effectué par une chondroïtinase et notamment par une chondroïtinase C ou par une chondroïtinase ABC qui fournit notamment les disaccharide 6-sulfurique, 4-sulfurique et 2,6-disulfurique, accompagnés de produits plus sulfatés. Des disaccharides non sulfatés présents dans toutes les origines sont également libérés par cette digestion enzymatique.Enzymatic cleavage can be performed by a chondroitinase and chondroitinase C or chondroitinase ABC which provides including 6-sulfuric, 4-sulfuric and 2,6-disulphuric disaccharide, accompanied by more sulphated products. Non-sulphated disaccharides present in all origins are also released by this enzymatic digestion.
L'analyse des chondroïtines sulfates peut être effectuée par RMN et montre des spectres différents qui sont la conséquence de la présence des différentes sulfatations et de leur proportion respective. De même, l'analyse par IRTF met en évidence dans la région située entre 1000 et 500 cm-1, et plus particulièrement entre 950 - 700 cm-1, des pics caractéristiques de chacun des acides chondroïtine sulfuriques. Les différentes chondroïtines d'origine porcine, ichtyque (requin) et bovine possèdent des spectres différents qui attestent de la présence et des proportions des différents disaccharides sulfuriques en quantités différentes.The analysis of the chondroitin sulphates can be carried out by NMR and shows different spectra which are the consequence of the presence of the different sulphatations and of their respective proportion. Likewise, the FTIR analysis shows in the region between 1000 and 500 cm -1 , and more particularly between 950 and 700 cm -1 , characteristic peaks of each of the chondroitin sulfuric acids. The various chondroitins of porcine, ichthyic (shark) and bovine origin have different spectra which attest to the presence and proportions of different sulfuric disaccharides in different quantities.
En outre, une réaction de β-élimination (entre le xylose et la sérine), réalisée sur les chondroïtines sulfates après extraction, permet d'évaluer le taux et la masse moléculaire des protéines qui resteraient liées malgré les processus de purification. La technique d'analyse utilisée ici est la chromatographie liquide sur colonne d'exclusion avec comme détecteur mais non exclusivement, un détecteur à indice de réfraction. Cette méthode renseigne sur la présence de protéines résiduelles liées.In addition, a β-elimination reaction (between xylose and serine), performed on chondroitin sulfates after extraction, used to evaluate the rate and mass protein that would remain bound despite purification processes. The analytical technique used here is liquid chromatography on an exclusion column with as a detector but not exclusively, a refractive index detector. This method provides information on the presence of bound residual proteins.
La quantité de dérivés 6-sulfuriques obtenue est dépendante comme pour les dérivés 4 sulfurique, de l'origine animale et du tissu d'extraction. Ainsi pour toutes les origines, mis à part le requin les valeurs sont toujours inférieures à celles du dérivé 4-sulfurique. Pour le requin, les dérivés sulfuriques représentent au minimum 40 % de la totalité des disaccharides. L'acide chondroïtine 2,6-disulfurique ne se trouve en pratique à des concentrations supérieures à 10 % que dans les mélanges d'origine ichtyque.The quantity of 6-sulfuric derivatives obtained is dependent as for 4 sulfuric derivatives, animal origin and extraction tissue. So for all origins, apart from the shark the values are always lower than those of the 4-sulfuric derivative. For the shark, sulfuric derivatives represent at least 40% of the all disaccharides. In practice, chondroitin 2,6-disulphuric acid is concentrations greater than 10% than in mixtures of fish origin.
Les acides chondroïtine sulfuriques ou disulfuriques résultant du clivage enzymatique des tissus cartilagineux jouent un rôle important dans les propriétés physiques et chimiques du cartilage. A concentrations élevées ils attirent l'eau dans les tissus de telle sorte que le collagène est placé sous tension, ce qui à son tour, contribue à la résistance mécanique du cartilage aux forces de compression. Ces molécules synthétisées par les chondrocytes et secrétées dans l'espace intercellulaire, sont liées aux hyaluronans et forment des complexes moléculaires de haut poids moléculaire.Chondroitin sulfuric or disulfuric acids resulting from cleavage enzymatic cartilage tissue plays an important role in physical properties and chemical cartilage. At high concentrations they attract water into the tissues of so that the collagen is put under tension, which in turn contributes to the resistance mechanical cartilage to compressive forces. These molecules synthesized by chondrocytes and secreted in the intercellular space, are related to hyaluronans and form molecular complexes of high molecular weight.
Dans les conditions physiologiques les chondrocytes articulaires sont responsables de l'évolution lente de la matrice extra cellulaire ce qui constitue une des causes des lésions des tissus.In physiological conditions the articular chondrocytes are responsible for the slow evolution of the extracellular matrix, which constitutes one of the causes of tissue damage.
Au cours du vieillissement, la taille de ces agrégats moléculaires, la teneur en acides chondroïtine sulfuriques, et la capacité de liaison des aggrécans tend à diminuer. Ces modifications peuvent être considérées comme un des facteurs qui interviennent dans la diminution de la capacité du cartilage à se réparer en cas de lésion et entraínent une prédisposition à des lésions ostéoarthritiques. Une fois le processus arthritique mis en place, les interleukines et notamment l'interleukine IL-1, jouent un rôle très important dans le métabolisme des aggrécans, en diminuant les processus de synthèse et en augmentant leur dégradation. La perte d'aggrécans conduit le réseau de collagène à une destruction mécanique et contribue à la progression de l'arthrose.During aging, the size of these molecular aggregates, the chondroitin sulfuric acid content, and the aggrecan binding capacity tends to decrease. These changes may be considered as one of the factors involved in reducing the ability of the cartilage to repair itself in case of injury and lead to a predisposition to osteoarthritic lesions. Once the process arthritis, interleukins, and especially interleukin IL-1, play a role in very important role in the metabolism of aggrecans, by decreasing the processes of synthesis and increasing their degradation. Aggression loss leads the network of collagen to mechanical destruction and contributes to the progression of osteoarthritis.
C'est pourquoi, les thérapeutiques qui manifestent des effets positifs "in vitro" sur l'anabolisme des aggrécans et/ou sur les capacités à empêcher les effets négatifs d'IL-1 sur le catabolisme des aggrécans sont susceptibles de retarder ou d'inhiber le processus de développement de la maladie ostéo-arthritique.This is why therapies that manifest positive effects "in in vitro "on the anabolism of aggrecans and / or the ability to prevent negative effects IL-1 on the catabolism of aggrecans are likely to delay or inhibit development process of osteoarthritic disease.
Les polysaccharides polysulfatés, et notamment ceux constitués de disaccharides disulfatés ont été utilisés chez des malades atteints d'osteo-arthrose et ont montré des résultats thérapeutiques importants, en particulier en ralentissant la progression de la maladie. Les disaccharides mono et/ou disulfatés peuvent être administrés par voie orale et peuvent atteindre des concentrations suffisantes dans les liquides biologiques - comme le liquide synovial- et dans les articulations pour exercer des effets bénéfiques.Polysulfated polysaccharides, and in particular those consisting of Disulfated disaccharides have been used in patients with osteoarthritis and have showed significant therapeutic results, especially by slowing down progression of the disease. Mono and / or disulfated disaccharides can be administered by and can reach sufficient concentrations in body fluids - like the synovial fluid- and in the joints to exert beneficial effects.
L'utilisation de molécules marquées au [35S] a montré des concentrations sériques importantes, et en particulier chez l'animal où l'on constate un tropisme sélectif vis-à-vis des tissus riches en GAG, tels que les disques intervertébraux, les yeux et le cartilage des articulations. D'une manière semblable, les acides chondroïtine mono- ou disulfatés manifestent un tropisme pour l'articulation du genou, ainsi qu'il a été mis en évidence par scanning (balayage) de l'os par CS marqué au 99 Tc.The use of [ 35 S] -labeled molecules has shown significant serum concentrations, and in particular in animals where there is a selective tropism towards GAG-rich tissues, such as intervertebral discs, the eyes and cartilage of the joints. Similarly, the mono- or disulfated chondroitin acids exhibit a tropism for the knee joint, as evidenced by scan (scanning) of the bone by 99 Tc-labeled CS.
Les acides chondroïtine mono- ou disulfuriques manifestent un effet protecteur sur l'endommagement du cartilage. Des études de dégradation du cartilage sur un modèle de lapin, induite par injection intra articulaire de chymopapaïne, ont montré un effet préventif sur la dégradation des cartilages. Les acides chondroïtine mono- ou disulfuriques manifestent "in vitro" des augmentations dans le taux de synthèses des aggrécans sur des chondrocytes humains et par là, contribuent à augmenter la teneur en protéines de la matrice, principalement les PG, dans l'environnement immédiat des chondrocytes, et de ce fait empêchent leur dédifférenciation.Chondroitin mono- or disulphuric acids have an effect protector on cartilage damage. Cartilage degradation studies on a rabbit model, induced by intra-articular injection of chymopapain, showed a preventive effect on cartilage degradation. Chondroitin mono- or disulfurics manifest "in vitro" increases in the synthesis rate of aggrecans on human chondrocytes and thereby help to increase the matrix proteins, mainly PGs, in the immediate environment of chondrocytes, and thus prevent their dedifferentiation.
Les études pharmacologiques effectuées avec les chondroïtines formées de disaccharides 2,4 ou 6-sulfuriques ainsi que de 2,6-disulfurique montrent une augmentation de la production et de la capacité d'aggrégation des protéoglycanes (PG) nouvellement synthétisés qui s'accumulent tout autour des chondrocytes mis en incubation dans des conditions de base. En outre, les acides chondroïtine mono- ou disulfuriques ont pour effet d'inverser au moins partiellement la désagrégation induite par IL-1 des protéoglycanes, et cet effet est observé "in vitro", même en l'absence de toute intervention biologique sur les cellules. Cet effet ne modifie pas le phénotype de chondrocyte comme il est mis en évidence par une expression stable de collagène de type II et d'aggrécan, sans hyper régulation du collagène de type I.Pharmacological studies performed with formed chondroitins 2,4- or 6-sulfuric disaccharides as well as 2,6-disulphuric acid increased production and aggregation ability of proteoglycans (PG) newly synthesized that accumulate all around the chondrocytes put in incubation under basic conditions. In addition, monounsaturated or disulphuric chondroitin have the effect of at least partially reversing the IL-1 induced disintegration of proteoglycans, and this effect is observed "in vitro" even in the absence of any intervention on cells. This effect does not alter the chondrocyte phenotype as it is demonstrated by stable expression of type II collagen and aggrecan, without hyper-regulation of type I collagen.
Cet effet n'est pas lié à la sulfatation de la molécule car l'héparine est inactive pour augmenter la synthèse des protéoglycanes.This effect is not related to sulfation of the molecule because heparin is inactive to increase proteoglycan synthesis.
De plus, les acides chondroïtine mono- et disulfatés augmentent la proportion de PG nouvellement synthétisés qui s'accumulent dans l'espace intercellulaire. Cette présence de PG en quantités importantes dans l'environnement des chondrocytes est un élément clé pour assurer la conservation du phénotype des chondrocytes et les propriétés physico-mécaniques du cartilage.In addition, mono- and disulfated chondroitin acids increase the proportion of newly synthesized PGs that accumulate in the intercellular space. This presence of PG in large quantities in the chondrocyte environment is key to ensuring the preservation of the chondrocyte phenotype and the physico-mechanical properties of the cartilage.
Les effets bénéfiques des acides chondroïtine mono- ou disulfuriques peuvent trouver une explication dans une augmentation de la synthèse de filaments plus longs d'hyaluronane, dans la quantité d'acide hyaluronique synthétisée et dans l'inhibition des activités enzymatiques, comme celle de l'élastase et de l'agrécanase ou de la cathepsine B. On peut donc supposer que les acides chondroïtine mono- ou disulfuriques peuvent avoir un effet sur la régulation des actions des métalloprotéases induites par IL-1 β et préservent ainsi le stock de PG non dégradés.The beneficial effects of chondroitin mono- or disulphuric acids can find an explanation in an increase in the synthesis of filaments more of hyaluronan, in the amount of hyaluronic acid synthesized and in the inhibition enzymatic activities, such as elastase and aggrecanase or cathepsin B. It can therefore be assumed that monounsaturated or disulphuric chondroitin have an effect on the regulation of metalloprotease actions induced by IL-1β and thus preserve the stock of non-degraded PGs.
Il apparaít principalement que les acides chondroïtine mono- et disulfuriques se distinguent par des interactions physico-chimiques avec les PG monomères. Un effet d'aggrégation similaire a été observé avec les acides chondroïtine sulfuriques lorsqu'ils sont mélangés avec des PG marqués au [35S], désagrégés juste avant d'être analysés par chromatographie. Cet effet est observé avec tous les types d'acide chondroïtine sulfurique, quelle que soit leur origine mais à des degrés divers. Par exemple, l'acide chondroïtine sulfurique extrait de cartilages de porc manifeste un pouvoir aggrégant puissant avec une augmentation des aggrégats de haut poids moléculaire (AHMW) de plus de 80 % manifesté par la mesure de radioactivité totale, alors que les acides extraits de cartilage d'autres espèces animales, telles que, le poulet ou le requin ont donné des augmentations de réaggrégation de 70 % en présence d'interleukine IL-1β par rapport à l'état normal.It appears mainly that mono- and disulphuric chondroitin acids are distinguished by physicochemical interactions with monomeric PGs. A similar aggregation effect was observed with chondroitin sulfuric acids when mixed with [ 35 S] labeled PGs, disaggregated just before being analyzed by chromatography. This effect is observed with all types of chondroitin sulfuric acid, regardless of their origin, but to varying degrees. For example, chondroitin sulfuric acid extracted from porcine cartilage exhibits potent aggregating power with an increase in high molecular weight aggregates (AHMW) of greater than 80% as measured by total radioactivity, whereas cartilage extracted acids other animal species, such as chicken or shark, gave 70% increase in reaggregation in the presence of interleukin IL-1β compared to the normal state.
Les acides chondroïtine mono- ou disulfuriques de l'invention augmentent le pourcentage de macromolécules sulfatées fonctionnelles dans l'environnement direct des chondrocytes "in vitro", principalement en manifestant des propriétés d'aggrégation des PG. Dans le cas où les cartilages sont endommagés, la rétention péri-cellulaire d'aggrécans volumineux, après induction par les acides chondroïtine mono- ou disulfuriques de l'invention, joue un rôle favorable pour retarder, ou mieux supprimer, le processus de dédifférenciation et pour amener une récupération d'un phénotype différencié.Chondroitin mono- or disulfuric acids of the invention increase the percentage of functional sulfated macromolecules in the direct environment of chondrocytes "in vitro", mainly by showing aggregation properties of PGs. In case the cartilages are damaged, the Peri-cellular retention of bulky aggrecans, after induction by the acids mono- or disulphuric chondroitin of the invention, plays a favorable role in retarding, or better to suppress, the process of dedifferentiation and to bring about a recovery of a differentiated phenotype.
Les acides chondroïtine mono (2,4 ou 6) sulfatés et l'acide chondroïtine 2,6-disulfurique de l'invention sont utilisés à des fins thérapeutiques, notamment par voie orale ou parentérale, sous forme de compositions pharmaceutiques telles que comprimés, dragées, gélules, pilules, cachets, poudres aromatisées ou non, ou d'injections destinées à la voie intramusculaire et intra-articulaire, sous forme d'ampoules, de fioles monodoses ou de seringue auto-injectables.Mono (2,4 or 6) sulfated chondroitin acids and chondroitin acid 2,6-disulfuric acid of the invention are used for therapeutic purposes, particularly by oral or parenteral, in the form of pharmaceutical compositions such as tablets, dragees, capsules, pills, cachets, powders, flavored or not, or injections intended for intramuscular and intra-articular, in the form of ampoules, single-dose vials or self-injecting syringe.
Les doses utilisées peuvent varier considérablement en fonction de l'ancienneté ou de l'évolution de la maladie. Les acides chondroïtine sulfuriques selon l'invention sont pratiquement dépourvus de toxicité et trouvent de ce fait un emploi très large en thérapeutique. Une dose usuelle pour la voie orale ou parentérale s'échelonne de 0,2 g à 2 g par prise unitaire et de préférence de 0,4 g à 0,6 g par prise.The doses used can vary considerably depending on seniority or evolution of the disease. Chondroitin sulfuric acids according to the invention are practically devoid of toxicity and thus find a very high employment wide in therapeutics. A usual dose for the oral or parenteral route ranges from 0.2 g to 2 g per unit dose and preferably 0.4 g to 0.6 g per dose.
Le tissu cartilagineux, en l'espèce les morceaux de trachée de bovins ou de porcins, est haché en petits fragments et soumis à une hydrolyse enzymatique en utilisant comme enzyme protéolytique l'enzyme commercialisé sous la dénomination Prolyve 100® par la société LYVEN. Il s'agit d'une protéase alcaline liquide, obtenue à partir d'une souche sélectionnée de Bacillus licheniformis. La fermentation est orientée pour produire essentiellement une protéase alcaline de type sérine, appelée aussi « subtilysine Carlsberg » (EC 3.4.21.14).Cartilaginous tissue, in this case the pieces of cattle trachea or pigs, is chopped into small pieces and subjected to enzymatic hydrolysis in using as the proteolytic enzyme the enzyme marketed under the name Prolyve 100® by the company LYVEN. It is a liquid alkaline protease, obtained at from a selected strain of Bacillus licheniformis. Fermentation is oriented to produce essentially an alkaline protease of the serine type, also called "Subtilysin Carlsberg" (EC 3.4.21.14).
Les chondroïtines sont séparées après filtration par addition d'éthanol sous agitation. Le précipité est essoré puis lavé par un mélange eau/ethanol. Le précipité est repris par un mélange eau/acétate de sodium, puis re-précipité par de l'éthanol sous agitation puis essoré, séché en étuve, broyé et tamisé sur maille de 450 µm.Chondroitins are separated after filtration by addition of ethanol with stirring. The precipitate is filtered off and then washed with a water / ethanol mixture. The precipitate is taken up in a water / sodium acetate mixture and then re-precipitated with ethanol under stirring and then drained, dried in an oven, milled and sieved on a mesh of 450 microns.
La chondroïtine ainsi obtenue est analysée en RMN du proton, ce qui permet de caractériser deux pics très aigus à 2,04 ppm (6S) et à 2,02 ppm (4S).The chondroitin thus obtained is analyzed by proton NMR, which characterizes two very sharp peaks at 2.04 ppm (6S) and 2.02 ppm (4S).
Les mêmes pics se retrouvent pour les chondroïtines d'origine ichtyque, bovine (essentiellement du 4 S) et porcine (pics à 2,02 et à 2,04 ppm). La chondroïtine d'origine ichtyque est la seule à fournir un pic important, correspondant à l'acide 6-chondroïtine sulfurique (voir figure 1).The same peaks are found for chondroitins of ichthyic origin, bovine (mainly 4S) and porcine (peaks at 2.02 and 2.04 ppm). Chondroitin of fish origin is the only one to provide a significant peak, corresponding to 6-chondroitin acid sulfuric acid (see Figure 1).
L'IRTF en pastilles de KBr montre entre 1 500 et 1 000 cm-1 les pics caractéristiques des chondroïtines de différentes origines (voir figure 2).The KBR pellet FTIR shows between 1500 and 1000 cm -1 the characteristic peaks of chondroitins of different origins (see Figure 2).
Des chondrocytes articulaires ont été mis en culture à une densité élevée pendant 6 jours. Les cellules sont incubées en présence ou en l'absence d'effecteurs à 10 ou 100 µg/ml en présence ou en l'absence d'IL-1β pendant 20 heures. Un transcript unique d'ARN de 8,9 kb et un transcript unique de procollagène II α 1 de 5 kb ont été observés dans des conditions de base.Joint chondrocytes were cultured at high density for 6 days. The cells are incubated in the presence or absence of 10-fold effectors. or 100 μg / ml in the presence or absence of IL-1β for 20 hours. A unique transcript 8.9 kb RNA and a single 5 kb procollagen II α 1 transcript were observed under basic conditions.
L'addition de 10 ou de 100 µg/ml d'acide chondroïtine sulfurique commercial (CS) n'a pas d'effet sur le rapport des ARNm aggrécan/GAPDH ou sur le rapport procollagène II α 1/GAPDH en comparaison avec des cellules non traitées.Addition of 10 or 100 mcg / ml commercial chondroitin sulfuric acid (CS) has no effect on the ratio of aggrecan mRNA / GAPDH or the ratio II procollagen α 1 / GAPDH compared with untreated cells.
En outre, aucune expression des produits de transcription (transcripts) de procollagène I 4,8 et 5,8 α 1 n'a été observée pendant que les fibroblastes cutanés étaient utilisés en tant que témoins positifs.In addition, no expression of transcription products (transcripts) of procollagen I 4.8 and 5.8 α 1 were observed while cutaneous fibroblasts were used as positive controls.
Le traitement des cellules avec IL-1 β a entraíné une diminution faible
mais significative des taux d'aggrécan et des taux d'ARNm de procollagène II α 1. L'ajout
de CS, n'a pas eu d'effet. Aucune expression d' ARNm de procollagène 1 n'a été détectable.
En résumé, le mode d'action des CS ne se situe vraisemblablement pas au niveau de la
modulation de la synthèse des ARNm du Col II ou des aggrécans.Treatment of cells with IL-1 β resulted in a small but significant decrease in aggrecan levels and RNA levels m procollagen α II 1. The addition of CS, had no effect. No expression of m RNA of procollagen 1 was detectable.
In summary, the mode of action of CS will likely not at the level of modulating mRNA synthesis of Col II or aggrécans.
L'action des CS sur la synthèse de PG a été étudiée par incorporation de (35SO4] dans des cultures de chondrocytes traitées par le CS. Des chondrocytes ont été mis en culture comme décrit ci-dessus. L'incubation avec ou sans effecteur a été réalisée dans du milieu DMEM (DULBECCO Modified Eagle Médium) dépourvu de sulfate auquel on a ajouté du sulfate marqué radioactivement. Dans les conditions de base, sans aucun effecteur, le niveau de PG sulfate (35SO4] total, extrait du milieu de culture (M), et du pool de cellules correspondantes (C) a été en moyenne (± SEM) de 3,42 dpm x 10-4 /10 µg d'ADN avec 36 % récupéré dans la couche de cellules. L'addition de CS n'a pas modifié sensiblement la quantité de PG sulfate [35SO4] secrété dans le milieu de culture alors qu'une augmentation importante a été observée dans le pool cellulaire. Le taux de PG sulfate [35SO4] mesuré dans le pool cellulaire a été augmenté de 68 % au-dessus des valeurs témoins (p < 0,01) en présence de 10 µg/ml de CS représentant ainsi 45 % du PG sulfate [35SO4] total. Aucun effet de stimulation supplémentaire n'a été observé lorsqu'on a ajouté CS à des doses plus élevées.The action of CSs on PG synthesis was investigated by incorporation of ( 35 SO 4 ) in chondrocyte cultures treated with CS.Chondrocytes were cultured as described above. Effector was performed in radio-labeled sulphate-free sulfate-free DMEM (DULBECCO Modified Eagle Medium) medium.In the basic conditions, without any effector, the total PG sulfate ( 35 SO 4 ) level, extracted from culture medium (M), and the corresponding cell pool (C) was on average (± SEM) of 3.42 dpm x 10 -4 / 10 μg of DNA with 36% recovered in the cell layer. addition of CS did not significantly alter the amount of PG sulfate [ 35 SO 4 ] secreted in the culture medium while a significant increase was observed in the cell pool The PG sulfate level [ 35 SO 4 ] measured in the cell pool was increased 68% above the values less (p <0.01) in the presence of 10 μg / ml of CS thus representing 45% of the PG sulfate [ 35 SO 4 ] total. No additional stimulatory effect was observed when CS was added at higher doses.
En présence de concentrations croissantes (2,10 ou 20 ng/ml) d'IL-1β
seul, le PG sulfate [35SO4] total récupéré dans le pool cellulaire a été notablement diminué,
d'une manière proportionnelle aux doses, représentant ainsi 37%, 10% et 6% de PG sulfate
[35SO4]. néo-synthétisé (Tableau 3). L'addition de CS à 10 ou 100 µg/ml avec 20 ng d'IL-1β
n'a pas modifié la quantité de PG sulfate [35SO4] secrété dans le milieu de culture alors
qu'une importante stimulation de PG [35SO4] sulfaté récupéré dans le pool cellulaire a été
observée.
CS a donc une action sur la synthèse des PG synthétisés par les chondrocytes.In the presence of increasing concentrations (2.10 or 20 ng / ml) of IL-1β alone, the total PG sulfate [ 35 SO 4 ] recovered in the cell pool was significantly decreased, in a dose-proportional manner, representing thus 37%, 10% and 6% PG sulfate [ 35 SO 4] . neo-synthesized (Table 3). The addition of CS at 10 or 100 μg / ml with 20 ng of IL-1β did not modify the amount of PG sulfate [ 35 SO 4] secreted in the culture medium while a significant stimulation of PG [ Sulfate SO 4 recovered in the cell pool was observed.
CS has an action on the synthesis of PGs synthesized by chondrocytes.
L'action des CS sur l'aggrégation des PG néosynthétisés a été investiguée. L'IL 1β a pour propriété de désagréger les PG ce qui se traduit lorsque l'on réalise une séparation par chromatographie d'exclusion des PG synthétisés par les chondrocytes, par une augmentation de complexes de plus bas poids moléculaires. Dans les conditions de base, plus de 80 % (± 5%) du PG sulfate [35SO4] ont été éluées sous forme de complexes agrégés de haut poids moléculaire (AHMW) (Kav = 0,10 - 0,40, zone en pointillé) avec moins de 10 % (± 3%) sous forme de PG non agrégé (Kav 0,40 à Kav 0,60).The action of CSs on the aggregation of neo-synthesized PGs has been investigated. The IL 1β has the property of disintegrating the PGs, which results when a separation by exclusion chromatography of the PGs synthesized by the chondrocytes is carried out, by an increase in complexes of lower molecular weight. Under the basic conditions, more than 80% (± 5%) of PG sulfate [ 35 SO 4 ] was eluted as aggregates of high molecular weight (AHMW) (K av = 0.10 - 0.40, dotted area) with less than 10% (± 3%) as non-aggregated PG (Ka v 0.40 to Ka v 0.60).
Dans les cellules traitées par IL-1β, une quantité décroissante d'une manière dose-dépendante, de PG aggrégée par (AHMW) a été observée en comparaison aux conditions témoin (figure 2B) représentant ainsi 73 (± 5%), 59 (± 6 %), et 38(± 5%) de la radioactivité totale sur la colonne avec 2, 10, et 20 ng/ml de IL-1β respectivement. La quantité de complexes agrégés AHMW récupérés dans les échantillons de PG sulfates [35SO4] à partir de cellules traitées par 20 ng/ml d'IL-1β et soit 10 ou 100 µg/ml de CS a été respectivement augmenté jusqu'à 60 % (± 4%) et ϒ (± 5%) (p < 0,01) en comparaison avec 38 % (± 6%) en présence de IL- 1β seul. In IL-1β-treated cells, a decreasing dose-dependently amount of PG agglomerated with (AHMW) was observed compared to the control conditions (FIG. 2B) thus representing 73 (± 5%), 59 ( ± 6%), and 38 (± 5%) of the total radioactivity on the column with 2, 10, and 20 ng / ml of IL-1β respectively. The amount of aggregated AHMW complexes recovered in the PG sulfate [ 35 SO 4 ] samples from cells treated with 20 ng / ml of IL-1β and either 10 or 100 μg / ml of CS was respectively increased up to 60% (± 4%) and Υ (± 5%) (p <0.01) compared to 38% (± 6%) in the presence of IL-1β alone.
Ces résultats sont la moyenne (±SD) de trois expériences séparées effectuées avec des chondrocytes provenant d'animaux différents.These results are the mean (± SD) of three separate experiments performed with chondrocytes from different animals.
Les CS sont donc capables de limiter la désagrégation de complexes de PG de haut poids moléculaire.CSs are therefore able to limit the disaggregation of complex PG of high molecular weight.
L'action des CS sur l'expression des MMP, protéases naturellement synthétisées par les chondrocytes, a été étudiées par "Northern Blot" après extraction des ARN de chondrocytes traités.The action of CS on the expression of MMPs, proteases naturally synthesized by chondrocytes, was studied by "Northern Blot" after extraction of RNA of chondrocytes treated.
L'expression d'ARNm de MMP - 1, -3 et - 1,3 a été augmentée fortement quand les cellules ont été traitées par IL-1β alors qu'aucun signal détectable n'a été observé dans les conditions témoin (DMEM dépourvu de sérum) ou en présence de CS (10 µg/ml) seul.MMP-1, -3, and -1.3 mRNA expression was increased strongly when the cells were treated with IL-1β whereas no detectable signal was observed under control conditions (DMEM devoid of serum) or in the presence of CS (10 μg / ml) alone.
L'addition simultanée de IL-1β et de CS aux cellules de la culture n'a pas modifié les taux d'ARNm des MMP.The simultaneous addition of IL-1β and CS to the cells of the culture did not modified MMP mRNA levels.
L'exposition des cellules à IL-1β plus CS (10 µg/ml) n'a pas modifié l'activité gélatinolytique principale observée dans les conditions basales d'un poids moléculaire apparent à 72 kDa (MMP-2), ni les activités supplémentaires observées en présence de IL-1β à un poids moléculaire apparent de 92 kDa et de 53/58 kDa, correspondant vraisemblablement à MMP-1,3 ou 9 et à MMP-1 ou 3 respectivement.Exposure of cells to IL-1β plus CS (10 μg / ml) did not change the main gelatinolytic activity observed under the basal conditions of a weight apparent molecular weight at 72 kDa (MMP-2), nor the additional activities observed in presence of IL-1β at an apparent molecular weight of 92 kDa and 53/58 kDa, probably corresponding to MMP-1,3 or 9 and MMP-1 or 3 respectively.
L'action des CS ne se situe pas au niveau de l'expression des MMPs investiguées dans ces expériences, ni au niveau de l'activité gélatinolytique dans les conditions expérimentales.The action of CS is not at the level of the expression of MMPs investigated in these experiments, nor at the level of gelatinolytic activity in the experimental conditions.
L'action de différentes sources de CS sur l'agrégation physique des PG synthétisés par les chondrocytes a ici été investiguée directement en mettant en contact les PG avec les CS. Des échantillons de quantités semblables de PG sulfates [35SO4] provenant de chondrocytes traités par 20 ng/ml d'IL-1β ont été mélangés avec des CS de différentes origines avant d'être appliqués sur la colonne de Sépharose.The action of different CS sources on the physical aggregation of PGs synthesized by chondrocytes has been investigated directly by putting PGs in contact with CSs. Samples of similar amounts of PG sulfates [ 35 SO 4 ] from chondrocytes treated with 20 ng / ml IL-1β were mixed with CSs of different origins before being applied to the Sepharose column.
Pour chaque profil d'élution, la quantité de PG agrégé éluée entre Kav 0,10 - 0,40 a été quantifiée et exprimée en tant que pourcentage de la radioactivité totale éluée sur la colonne. For each elution profile, the amount of aggregated PG eluted between Kav 0.10 - 0.40 was quantified and expressed as a percentage of total radioactivity eluted on the column.
Les résultats figurent dans le tableau 3.The results are shown in Table 3.
Alors qu'en présence d'IL-1β (20 ng/ml) seul, 49 ± 6 % du PG [35S] sulfaté ont été élués sous forme de PG agrégés par AHMW (Kav 0,1 - 0,4), l'addition de chaque polymère a augmenté notablement la proportion de molécules agrégées AHMW jusqu'à plus de 70 % de la radioactivité totale. Le pouvoir d'aggrégation maximale (plus de 80 % de la radioactivité totale) a été observé avec des CS d'origine porcine (CS 204, CS-206 et CS-IG 336) alors que ceux d'autres origines étaient moins actives. Très clairement, selon la source de CS, une action de réagrégation plus ou moins prononcée a été mise en évidence dans ces conditions expérimentales.Whereas in the presence of IL-1β (20 ng / ml) alone, 49 ± 6% of the sulfated PG [ 35 S] was eluted as AHMW-aggregated PG (K av 0.1 - 0.4) the addition of each polymer significantly increased the proportion of aggregated AHMW molecules to more than 70% of the total radioactivity. The maximum aggregation power (more than 80% of total radioactivity) was observed with CS of porcine origin (CS 204, CS-206 and CS-IG 336) while those of other origins were less active. Very clearly, according to the source of CS, a more or less pronounced re-aggregation action has been demonstrated under these experimental conditions.
Acide chondroïtine dsulfurique d'origine porcine contenant environ 60 % d'acide
chondroïtine, 4-sulfurique 500 g,
Lactose 50 g,
Stéarate de Magnésium 20 g
pour 1.000 gélules d'un poids moyen de 0,55 g
Colorant jaune qs
Lactose 50 g,
Magnesium Stearate 20 g
per 1,000 capsules with an average weight of 0.55 g
Yellow dye qs
Effet de CS sur l'incorporation totale de [35SO4] sulfate dans les protéoglycanes
néosynthétisés
Claims (11)
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| FR0403020A FR2867981B1 (en) | 2004-03-24 | 2004-03-24 | USE OF MONO-AND DISULFATE CHONDROITINE IN THERAPEUTICS |
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| RU2080857C1 (en) * | 1994-10-07 | 1997-06-10 | Научно-производственное объединение "Иммунопрепарат" | Method of preparing the preparation chondroitin sulfate injection form |
| RU2200018C1 (en) * | 2001-12-26 | 2003-03-10 | Государственный институт кровезаменителей и медицинских препаратов | Method of preparing injection form of preparation chondroitin sulfate |
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| US5498608A (en) | 1994-01-07 | 1996-03-12 | Salix Pharmaceuticals | Use of 2-hydroxy-5-phenylazobenzoic acid derivatives as colon cancer chemopreventative and chemotherapeutic agents |
| WO2001093838A1 (en) | 2000-06-06 | 2001-12-13 | Nutratech, Inc. | Sodium-free chondroitin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITMI20121316A1 (en) * | 2012-07-27 | 2014-01-28 | Altergon Sa | CONDROTIC COMPLEXES FOR TRANSCUTANEOUS ABSORPTION |
| WO2014016380A1 (en) * | 2012-07-27 | 2014-01-30 | Altergon S.A. | Chondroitin complexes for transcutaneous absorption |
| CN104684542A (en) * | 2012-07-27 | 2015-06-03 | 阿尔特刚股份有限公司 | Chondroitin complexes for transcutaneous absorption |
| EP2877156B1 (en) * | 2012-07-27 | 2020-01-01 | Altergon S.a. | Chondroitin complexes for transcutaneous absorption |
| US10675270B2 (en) | 2012-07-27 | 2020-06-09 | Altergon S.A. | Chondroitin complexes for transcutaneous absorption |
Also Published As
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| FR2867981A1 (en) | 2005-09-30 |
| EP1582214A3 (en) | 2005-10-12 |
| FR2867981B1 (en) | 2008-10-31 |
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